Combined administration of integrin receptor antagonists for anti-angiogenic therapy

ABSTRACT

The teachings provided herein generally relate to a combination therapy and are directed to pharmaceutical compositions and methods for administering a combination of an αvβ3 antagonist with an α2β1 antagonist to a subject. The methods are for use in inhibiting, preventing, or reversing angiogenesis, as well as in treating cancer. In some embodiments, the compositions and methods include a combined administration of echistatin and VP12 (ECL12).

BACKGROUND

1. Field of the Invention

The teachings provided herein relate to pharmaceutical compositionscomprising an αvβ3 antagonist for use in inhibiting angiogenesis andtreating cancer when used in combination with an α2β1 antagonist and ina pharmaceutically acceptable carrier.

2. Description of Related Art

Solid tumor growth is generally considered to be angiogenesis-dependent,such that the control of neovascularization in cancerous tissue is oneof the goals of cancer research. As such, various potential angiogenesisinhibitors have been investigated in the treatment of solid tumors andmetastasis using anti-angiogenic therapy. Unfortunately, however, aparticularly effective method of using an agent or combination of agentsremain to be discovered that, at least, (i) inhibits or preventsangiogenesis; (ii) treats solid tumors to contain and/or reduce tumorsize; and (iii) inhibits or prevents the tumor invasion that leads tometastasis within a subject.

The art is still in need of improved angiogenesis inhibitors, as wellcancer therapies that include such inhibitors. Angiogenesis is a highlyregulated event that involves complex, dynamic interactions betweenmicrovascular endothelial cells and extracellular matrix (ECM) proteins.Control of angiogenesis can be used in a variety of treatments,including cancer therapy. Alteration of ECM composition and architectureis a hallmark of wound clot and tumor stroma. The role of ECM inregulation of angiogenesis associated with wound healing and tumorgrowth still remain generally undefined in the art. During angiogenesis,however, endothelial cell responses to growth factors are modulated bythe compositional and mechanical properties of a surroundingthree-dimensional (3D) extracellular matrix (ECM) that is dominated byeither cross-linked fibrin or type I collagen.

Likewise, a novel method to control tumor invasion to treat a metastaticdisease, for example, would be seen as a significant contribution to theart by one of skill. Over 60% of breast cancer patients have metastaticdisease at diagnosis. The most common cause of death in breast cancerpatients is due to the metastatic spread of the cancer cells from theprimary tumor site to remote sites and growth of the breast cancer cellsat the distant location. Metastasis is a complex process includingseveral mechanisms: (1) migration of the tumor cells through theextracellular matrix surrounding the tumor; (2) invasion of tumor cellsinto angiogenic blood vessels growing into the tumor; (3) adhesion ofthe metastatic cell at a distant site where the microenvironment isreceptive to tumor growth; and (4) newly attached cells must proliferateand induce angiogenesis at the metastatic site. As such, a combinationof select inhibitors could possibly limit this process.

Accordingly, and for at least the above reasons, one of skill willappreciate a method of inhibiting, preventing, or even reversing,angiogenesis. Moreover, one of skill will appreciate a composition andmethod of treatment that can not only inhibit angiogenesis, but that canalso disrupt the physical and mechanical architecture within whichangiogenesis takes place. Such a composition and method may be able to,at least, (i) inhibit or prevent angiogenesis; (ii) treat solid tumorsto contain and/or reduce tumor size; and (iii) inhibit or prevent thetumor invasion that leads to metastasis within a subject.

SUMMARY

The teachings provided herein generally relate to pharmaceuticalcompositions and methods comprising an αvβ3 antagonist for use ininhibiting, preventing, or reversing angiogenesis and treating cancerwhen used in combination with an α2β1 antagonist and in apharmaceutically acceptable carrier. In some embodiments, the teachingsare directed to a pharmaceutical formulation comprising an αvβ3antagonist, an α2β1 antagonist, and a pharmaceutically acceptablecarrier. The pharmaceutical formulation can, for example, compriseechistatin and VP12 (ECL12). In some embodiments, the teachings aredirected to an article of manufacture comprising an αvβ3 antagonist, anα2β1 antagonist, and instructions for administering an effective amountof the αvβ3 antagonist and an effective amount of the α2β1 antagonist toa subject.

In some embodiments, the methods are directed to inhibiting angiogenesisin a subject, comprising administering an effective amount of an αvβ3antagonist in combination with an effective amount of an α2β1 antagonistto the subject. The methods can comprise, for example, administering andeffective amount of echistatin and VP12 (ECL12) to the subject. In someembodiments, the method further inhibits tumor invasion. And, in someembodiments, the method can inhibit the growth of solid tumors.

In some embodiments, the methods taught herein can further include theadministration of an effective amount of an antiproliferative. And, insome embodiments, the methods can include the administration of aneffective amount of radiation therapy.

In some embodiments, the methods can be directed to inhibitingangiogenesis, inhibiting tumor invasion, inhibiting the growth of solidtumors, or a combination thereof, in a subject. In these embodiments, asdescribed above, the methods can further comprise the administration ofan effective amount of an antiproliferative, an effective amount ofradiation therapy, surgical therapy, or a combination thereof.

One of skill reading the teachings that follow will appreciate that theconcepts can extend into additional embodiments that go well-beyond aliteral reading of the claims, the inventions recited by the claims, andthe terms recited in the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a study of human microvascular endothelial cellangiogenesis, according to some embodiments.

FIGS. 2A-2D show the HDMEC of the model with angiogenesis formation,according to some embodiments.

FIGS. 3A-3F show would repair in 5, 7, and 10 day porcine wounds,according to some embodiments.

FIGS. 4A-4F show the staining of 5, 7, and 10 day wound specimens withMasson trichrome, according to some embodiments.

FIGS. 5A-5E show that fibrin supports sprouting angiogenesis, accordingto some embodiments.

FIGS. 6A-6H show that fibrin supports sprouting angiogenesis wherecollagen does not support sprouting angiogenesis, according to someembodiments.

FIGS. 7A-7D show synergistic inhibition of sprout angiogenesis whencombining echistatin, an αvβ3 antagonist with VP12 (ECL12), an α2β1agonist, according to some embodiments.

DETAILED DESCRIPTION

The teachings provided herein generally relate to pharmaceuticalcompositions and methods comprising an αvβ3 antagonist for use ininhibiting, preventing, or reversing angiogenesis and treating cancerwhen used in combination with an α2β1 antagonist and in apharmaceutically acceptable carrier.

In some embodiments, the teachings are directed to a pharmaceuticalformulation comprising an αvβ3 antagonist, an α2β1 antagonist, and apharmaceutically acceptable carrier. In some embodiments, the teachingsare directed to an article of manufacture comprising an αvβ3 antagonist,an α2β1 antagonist, and instructions for administering an effectiveamount of the αvβ3 antagonist and an effective amount of the α2β1antagonist to a subject. In some embodiments, the terms “composition”and “formulation” can be interchangeable.

In some embodiments, the teachings are directed methods of inhibitingangiogenesis in a subject, comprising administering an effective amountof an αvβ3 antagonist in combination with an effective amount of an α2β1antagonist to the subject. In some embodiments, the method furtherinhibits tumor invasion. And, in some embodiments, the method caninhibit the growth of solid tumors.

The αvβ3 antagonist can include, for example, any chemical moiety thatfunctions to block αvβ3. Likewise, the α2β1 antagonist can include, forexample, any chemical moiety that functions to block α2β1. Suchantagonists can include small molecules, such as small moleculepharmaceuticals, or large molecules, such as peptides, oligopeptides,polypeptides, proteins, nucleic acids, oligonucleotides, andpolynucleotides, for example. In some embodiments, the peptide caninclude an RGD-recognition motif. In some embodiments, an antagonist caninclude an antibody such as, for example, a polyclonal antibody or amonoclonal antibody and, in some embodiments, the antibody can behumanized or fully human. The antibody may already be known to bind toan αvβ3 antagonist, an α2β1 antagonist, or a combination thereof; or,the antibody can be designed specifically to bind to an αvβ3 antagonist,an α2β1 antagonist, or a combination thereof. One of skill will how toselect and/or design an antibody of interest for use with the methodsprovided herein and can appreciate, for example, that there are knownmethods of producing a desired antibody. As such, any inhibitor, orligand, that binds to, and down-regulates, the activity of angiogenesisand/or collagen matrix formation can be used in some embodiments. Suchinhibitors can include, but are not limited to, disintegrins, RGDpeptides, blocking monoclonal antibodies, chemical inhibitors, antisensemRNA, and the like, or any combination thereof.

In some embodiments, the antagonists can include one or more of thedisintegrins that bind to an αvβ3 antagonist, an α2β1 antagonist, or acombination thereof. Examples of disintegrins can include disintegrinsobtained from snake venom extracts. Such integrins can include, forexample, RGD and non-RGD, such as KGD, MLD, VGD, and MVD disintegrins,referring to an active peptide sequence which can be, for example, inthe “inhibitory loop” of the sequence.

The disintegrins from snake venom can comprise (i) a first group ofsingle chain sequence compounds having about 49-51 residues and fourdisulphide bonds; (ii) a second group of single chain sequence compoundshaving about 70 residues and six disulphide bonds; (iii) a third groupof single chain sequence compounds having about 84 residues cross-linkedby seven disulphide bonds; (iv) a fourth group of single chain sequencecompounds having about 100 residues having 16 Cys residues involved informing eight disulphide bonds; and (v) a fifth group of dimericcompounds having homodimers or heterodimers. The dimeric disintegrinscan contain, for example, about 67 residues in each subunit, with tencysteine residues involved in forming four intrachain disulphide bondsand two interchain cysteine linkages.

In some embodiments, the disintegrins can include echistatin, VLO4, VP12(ECL12), or a combination thereof. In some embodiments the disintegrinscan comprise (i) echistatin, eristocophin, eristostatin, andocellatusin; (ii) trigramin, kistrin, flavoridin, albolabrin, andbarbourin; (iii) bitistatin and salmosin 3; (iv) PIII; and (v)contortrostatin, EC3, bilitoxin, and EMF-10; and a combination thereof.And, in some embodiments, the disintegrins can include, for example,EO4, EO5, EMS11, VLO4, VLO5, VB7, VA6, or a combination thereof.

In some embodiments, the methods can comprise, for example,administering and effective amount of echistatin and VP12 (ECL12) to thesubject. In some embodiments, contortrostatin may be used in place of,or in addition to, VP12 (ECL12). In some embodiments, an RGD peptidethat blocks αvβ3 integrin activity can be used in combination with amonoclonal antibody that blocks α2β1 integrin activity.

The antagonists include proteins, such as the disintegrins. And, anypolypeptide having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% identity to one of the proteins taught herein can be used.The term “identity” can be used to refer to the extent to whichsequences are invariant. The identity can be referenced against anentire protein or a defined fragment of the protein, as well as, perhapscombination of protein fragments in the case of a construct of peptidefragments. Computational approaches to sequence alignment are generaleither global or local alignments. Calculating a global alignment is aform of global optimization that “forces” the alignment to span theentire length of all query sequences. By contrast, local alignments canbe used to identify regions of similarity within long sequences that areoften widely divergent overall. Local alignments can be used, but can bemore difficult to calculate because of the additional challenge ofidentifying the regions of similarity. One of skill will appreciate thata variety of computational algorithms are available, including slow butformally optimizing methods like dynamic programming, and efficient, butnot as thorough heuristic algorithms or probabilistic methods designedfor large-scale database search. In some embodiments, the sequencealignments for identity can be local, global, dynamic, progressive,heuristic or probabilistic. And, in some embodiments, the sequencealignments can even be based around the location of a motif of interest,such as an active region of the protein sought for binding. The motif inthe disintegrins, for example, can include the inhibitory loop region,an RGD region, a comparable non-RGD active region, or a combinationthereof. One of skill can readily compare sequence identity for any of anumber of desired purposes such as, for example, assessing thepossibility of binding to a receptor, functioning as an agonist,antagonist, and the like, given the knowledge of the function of a likeprotein or peptide structure. Examples of programs that can be used todetermine identity or homology include, for example, BLAST and FASTA.

A polypeptide can include a variant or mutant of the protein, a chimericconstruct, a fragment, a construct of at least two linked peptidefragments, variants of the fragments, a dimer, and the like. Any of thepolypeptides taught herein can be produced using recombinant proceduresknown to one of skill, or using synthetic procedures in which the aminoacid sequence can be constructed, for example, using liquid or solidphase synthesis techniques, such as Fmoc or Boc methods. Likewise, anyof the polypeptides taught herein can be isolated and/or purified usingprocedures known to one of skill, such as through the use of affinitytags, and the like. In some embodiments, the polypeptides can includethe inhibitory loop regions, RGD or non-RGD peptide fragments, linkersthat include an amino acid, an amino acid sequence, alkylenes, or acombination thereof.

The term “variant” refers to modifications to a peptide that allows thepeptide to retain its binding properties, and such modificationsinclude, but are not limited to, conservative substitutions in which oneor more amino acids are substituted for other amino acids; deletion oraddition of amino acids that have minimal influence on the bindingproperties or secondary structure; conjugation of a linker;post-translational modifications such as, for example, the addition offunctional groups. Examples of such post-translational modifications caninclude, but are not limited to, the addition of modifying groupsdescribed below through processes such as, for example, glycosylation,acetylation, phosphorylation, modifications with fatty acids, formationof disulfide bonds between peptides, biotinylation, PEGylation, andcombinations thereof.

In many embodiments, the molecular weight of an agent should be at orbelow about 40,000 Daltons to ensure elimination of the agent from asubject. In some embodiments, the molecular weight of the agent rangesfrom about 300 Daltons to about 40,000 Daltons, from about 8,000 Daltonsto about 30,000 Daltons, from about 10,000 Daltons to about 20,000Daltons, or any range therein.

The variants can be merely conservatively modified variants of thepolypeptides containing only conservative substitutions. The term“conservatively modified variant” refers to a conservative amino acidsubstitution, which is an amino acid substituted by an amino acid ofsimilar charge density, hydrophilicity/hydrophobicity, size, and/orconfiguration such as, for example, substituting valine for isoleucine.In comparison, a “non-conservatively modified variant” refers to anon-conservative amino acid substitution, which is an amino acidsubstituted by an amino acid of differing charge density,hydrophilicity/hydrophobicity, size, and/or configuration such as, forexample, substituting valine for phenyalanine.

In some embodiments, the methods taught herein can further include theadministration of an effective amount of an additional bioactive agentor therapeutic treatment, such as the administration of an effectiveamount of an antiproliferative and/or an effective amount of radiationtherapy. In some embodiments, the terms “agent” and “therapy” can beinterchangeable. For example, the administration of radiation can beconsidered the administration of a second agent, in some embodiments.

A bioactive agent can be any moiety capable of contributing to atherapeutic effect, a prophylactic effect, both a therapeutic andprophylactic effect, or other biologically active effect in a subject. Abioactive agent can also have diagnostic properties. The bioactiveagents include, but are not limited to, small molecules, nucleotides,oligonucleotides, polynucleotides, amino acids, oligopeptides,polypeptides, and proteins. Bioactive agents can include, but are notlimited to, antiproliferatives, antineoplastics, antimitotics,anti-inflammatories, antiplatelets, anticoagulants, antifibrins,antithrombins, antibiotics, antiallergics, antioxidants, and anyprodrugs, codrugs, metabolites, analogs, homologues, congeners,derivatives, salts and combinations thereof. It is to be appreciatedthat one skilled in the art should recognize that some of the groups,subgroups, and individual bioactive agents may not be used in someembodiments of the present invention.

Antiproliferatives include, for example, actinomycin D, actinomycin IV,actinomycin I1, actinomycin X1, actinomycin C1, and dactinomycin(Cosmegen®, Merck & Co., Inc.). Antineoplastics or antimitotics include,for example, paclitaxel (TAXOL, Bristol-Myers Squibb Co.), docetaxel(TAXOTERE, Aventis S.A.), methotrexate, azathioprine, vincristine,vinblastine, fluorouracil, doxorubicin hydrochloride (ADRIAMYCIN,Pfizer, Inc.) and mitomycin (MUTAMYCIN, Bristol-Myers Squibb Co.), andany prodrugs, codrugs, metabolites, analogs, homologues, congeners,derivatives, salts and combinations thereof. Antiplatelets,anticoagulants, antifibrin, and antithrombins include, for example,sodium heparin, low molecular weight heparins, heparinoids, hirudin,argatroban, forskolin, vapiprost, prostacyclin and prostacyclinanalogues, dextran, D-phe-pro-arg-chloromethylketone (syntheticantithrombin), dipyridamole, glycoprotein IIb/IIIa platelet membranereceptor antagonist antibody, recombinant hirudin, and thrombininhibitors (ANGIOMAX, Biogen, Inc.), and any prodrugs, codrugs,metabolites, analogs, homologues, congeners, derivatives, salts andcombinations thereof. Cytostatic or antiproliferative agents include,for example, angiopeptin, angiotensin converting enzyme inhibitors suchas captopril (CAPOTEN and CAPOZIDE, Bristol-Myers Squibb Co.),cilazapril or lisinopril (PRINVIL and PRINZIDE, Merck & Co., Inc.);calcium channel blockers such as nifedipine; colchicines; fibroblastgrowth factor (FGF) antagonists, fish oil (omega 3-fatty acid);histamine antagonists; lovastatin (MEVACOR, Merck & Co., Inc.);monoclonal antibodies including, but not limited to, antibodies specificfor Platelet-Derived Growth Factor (PDGF) receptors; nitroprusside;phosphodiesterase inhibitors; prostaglandin inhibitors; suramin;serotonin blockers; steroids; thioprotease inhibitors; PDGF antagonistsincluding, but not limited to, triazolopyrimidine; and nitric oxide, andany prodrugs, codrugs, metabolites, analogs, homologues, congeners,derivatives, salts and combinations thereof. Antiallergic agentsinclude, but are not limited to, pemirolast potassium (ALAMAST, Santen,Inc.), and any prodrugs, codrugs, metabolites, analogs, homologues,congeners, derivatives, salts and combinations thereof.

Antibody therapy provides additional bioactive agents that may be usefulwhen administered in combination with the methods taught herein.AVASTATIN, for example, is a human monoclonal antibody to VEGF, hasprovided beneficial results in colorectal cancer, increasing survivaltime by more than 30% when used in combination with the standard Saltzregime of irinotecan, 5-fluorouracil, and leucovorin. One of skill willappreciate that several monoclonal antibodies would be useful, thefollowing providing further examples:

TABLE mAb name Trade name Cancer treated: rituximab RITUXAN non-Hodgkinlymphoma trastuzumab HERCEPTIN breast cancer gemtuzumab MYLOTARG acutemyelogenous ozogamicin* leukemia (AML) alemtuzumab CAMPATH chroniclymphocytic leukemia (CLL) ibritumomab ZEVALIN non-Hodgkin lymphomatiuxetan* tositumomab* BEXXAR non-Hodgkin lymphoma cetuximab ERBITUXcolorectal cancer; head & neck cancers bevacizumab AVASTIN colorectalcancer; non-small cell lung cancer; breast cancer; glioblastoma; kidneycancer panitumumab VECTIBIX colorectal cancer ofatumumab ARZERRA chroniclymphocytic leukemia (CLL) *refers to a conjugated monoclonal antibody

It should be appreciated that, a bioactive agent can be given alone orin combination with other bioactive agents, with the compositions andmethods taught herein. Chemotherapy drugs, for example, are sometimesmost effective when given in combination, as a combination chemotherapyregime. The rationale for combination chemotherapy is to use drugs thatwork by different mechanisms of action, thereby decreasing thelikelihood that resistant cancer cells will develop. When drugs havingdifferent effects are combined, each drug can be used at its optimaldose, sometimes without, and sometimes reducing, intolerable sideeffects.

For some cancers, the best approach may be a combination of surgery,radiation therapy, and/or chemotherapy. Surgery or radiation therapy,for example, treats cancer that is confined locally, while chemotherapycan be used to also kill the cancer cells that have spread to distantsites. Sometimes radiation therapy or chemotherapy can be given beforesurgery to shrink a tumor, thereby improving the opportunity forcomplete surgical removal, making these types of combination therapies apotentially valuable therapy for use with the teachings provided herein,at least in some embodiments. Radiation therapy and low-dosechemotherapy after surgery, for example, can help destroy remainingcancer cells. One of skill will appreciate that the stage of the cancercan be a considerable factor in determining whether single therapy or acombination is desired. For example, early-stage breast cancer may betreated with surgery alone, or by using surgery combined with radiationtherapy, chemotherapy, or a combination thereof, depending on the sizeof the tumor and the risk of recurrence. Locally advanced breast cancer,for example, can be treated with chemotherapy, radiation therapy, andsurgery, in some embodiments.

Any cancer tissue that relies, at least in part, on blood supply tosurvive, may be treatable in some embodiments. Examples of cancers thatmay be treated using the methods taught herein can include, but are notlimited to, prostate, bladder, lung, breast, osteosarcoma, pancreatic,colon, melanoma, testicular, colorectal, urothelial, renal cell,hepatocellular, leukemia, lymphoma, and ovarian cancer and centralnervous system malignancies. Lung cancer, although often disperse, mayalso be treated in some embodiments. Likewise, even liquid cancers, suchas lymphoma and leukemia, including acute myeloid leukemia, may also betreated in some embodiments using methods that incorporate the teachingsprovided herein.

Sometimes the combinations taught herein may not be directed to a curebut, rather, to reduce symptoms and prolong life. Such combinationtherapies can be useful, for example, for subjects having advancedcancers that are not suitable for radiation therapy or surgicaltreatment, such as those having un-resectable non-small cell lungcancer, esophageal cancer, or bladder cancers, for example.

In some embodiments, the methods can comprise, for example,administering an effective amount of echistatin and an effective amountof VP12 (ECL12) to the subject, wherein a cytotoxic agent, such asTAXOL, G-CSF, or a combination thereof, for example, can be administeredto provide a combination therapy. And, in some embodiments, thesemethods can be accompanied by radiation therapy, surgical therapy, or acombination thereof.

In some embodiments, the methods can be directed to inhibiting orpreventing angiogenesis, reversing angiogenesis, inhibiting orpreventing tumor invasion, inhibiting or preventing the growth of solidtumors, reducing the size of solid tumors, or a combination thereof, ina subject. In these embodiments, as described above, the methods canfurther comprise the administration of an effective amount of anantiproliferative, an effective amount of radiation therapy, surgicaltherapy, or a combination thereof.

One of skill will appreciate that “tumor invasion” can be defined as thepenetration of tissue barriers by migrating cancerous cells, and tumorinvasion can be integral to metastases. Tumor invasion can include theinvasion of surrounding tissue as the tumor grows, and capillaryendothelial cells invade the tumor and create tumor blood vesselsthrough, i.e., neovascularization of the tumor tissue. The tumor cellscan also intravasate into the blood circulation for metastasis. Tumorcells can then arrest into distant organs, extravasate, and againmigrate into the new site and start the invasive cycle again. As such,the inhibition or prevention of tumor invasion can contain a tumor ortumors and inhibit or prevent metastasis.

Uses and Methods of Administration

The compositions can provide a therapeutic and/or prophylactic effect inthe treatment of a disease, or ameliorization of one or more symptoms ofa disease in a subject. The term “subject” and “patient” are usedinterchangeably and refer to an animal such as a mammal including, butnot limited to, non-primates such as, for example, a cow, pig, horse,cat, dog, rat and mouse; and primates such as, for example, a monkey ora human.

The compositions provided herein can be administered to a subject usingany manner of administration known to one of skill. For example, in someembodiments, a localized administration is used and, in some embodimentsa systemic administration is used. In some embodiments, a combination ofsystem and local administration is used. One of skill will appreciatethat the therapeutic program selected, the agents administered, thecondition of the subject, and the effects desired, can affect theadministration schedule and program used.

One of skill understands that the amount of the agents administered canvary according to factors such as, for example, the type of disease,age, sex, and weight of the subject, as well as the method ofadministration. For example, local and systemic administration can callfor substantially different amounts to be effective. Dosage regimens mayalso be adjusted to optimize a therapeutic response. In someembodiments, a single bolus may be administered; several divided dosesmay be administered over time; the dose may be proportionally reduced orincreased; or, any combination thereof, as indicated by the exigenciesof the therapeutic situation and factors known one of skill in the art.It is to be noted that dosage values may vary with the severity of thecondition to be alleviated. Dosage regimens may be adjusted over timeaccording to the individual need and the professional judgment of theperson administering or supervising the administration of thecompositions, and the dosage ranges set forth herein are exemplary onlyand do not limit the dosage ranges that may be selected by medicalpractitioners.

The terms “administration” or “administering” refer to a method ofincorporating a composition into the cells or tissues of a subject,either in vivo or ex vivo to diagnose, prevent, treat, or ameliorate asymptom of a disease. In one example, a compound can be administered toa subject in vivo parenterally. In another example, a compound can beadministered to a subject by combining the compound with cell tissuefrom the subject ex vivo for purposes that include, but are not limitedto, assays for determining utility and efficacy of a composition. Whenthe compound is incorporated in the subject in combination with one oractive agents, the terms “administration” or “administering” can includesequential or concurrent incorporation of the compound with the otheragents such as, for example, any agent described above. A pharmaceuticalcomposition of the invention is formulated to be compatible with itsintended route of administration. Examples of routes of administrationinclude, but are not limited to, parenteral such as, for example,intravenous, intradermal, intramuscular, and subcutaneous injection;oral; inhalation; intranasal; transdermal; transmucosal; and rectaladministration.

An “effective amount” of a compound of the invention can be used todescribe a therapeutically effective amount or a prophylacticallyeffective amount. An effective amount can also be an amount thatameliorates the symptoms of a disease. A “therapeutically effectiveamount” refers to an amount that is effective at the dosages and periodsof time necessary to achieve a desired therapeutic result and may alsorefer to an amount of active compound, prodrug or pharmaceutical agentthat elicits any biological or medicinal response in a tissue, system,or subject that is sought by a researcher, veterinarian, medical doctoror other clinician that may be part of a treatment plan leading to adesired effect. In some embodiments, the therapeutically effectiveamount may need to be administered in an amount sufficient to result inamelioration of one or more symptoms of a disorder, prevention of theadvancement of a disorder, or regression of a disorder. In someembodiments, for example, a therapeutically effective amount can referto the amount of an agent that provides a measurable response of atleast 5%, at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or at least 100% ofa desired action of the composition. The term “treating” refers to theadministering one or more therapeutic or prophylactic agents taughtherein.

A “prophylactically effective amount” refers to an amount that iseffective at the dosages and periods of time necessary to achieve adesired prophylactic result such as, preventing, inhibiting, orreversing angiogenesis, tumor growth, or tumor invasion. Typically, aprophylactic dose is used in a subject prior to the onset of a disease,or at an early stage of the onset of a disease, to prevent or inhibitonset of the disease or symptoms of the disease. A prophylacticallyeffective amount may be less than, greater than, or equal to atherapeutically effective amount.

The administration can be local or systemic. In some embodiments, theadministration can be oral. In other embodiments, the administration canbe subcutaneous injection. In other embodiments, the administration canbe intravenous injection using a sterile isotonic aqueous buffer. Inanother embodiment, the administration can include a solubilizing agentand a local anesthetic such as lignocaine to ease discomfort at the siteof injection. In other embodiments, the administrations may beparenteral to obtain, for example, ease and uniformity ofadministration.

The compounds can be administered in dosage units. The term “dosageunit” refers to discrete, predetermined quantities of a compound thatcan be administered as unitary dosages to a subject. A predeterminedquantity of active compound can be selected to produce a desiredtherapeutic effect and can be administered with a pharmaceuticallyacceptable carrier. The predetermined quantity in each unit dosage candepend on factors that include, but are not limited to, (a) the uniquecharacteristics of the active compound and the particular therapeuticeffect to be achieved, and (b) the limitations inherent in the art ofcreating and administering such dosage units.

A “pharmaceutically acceptable carrier” is a diluent, adjuvant,excipient, or vehicle with which the composition is administered. Acarrier is pharmaceutically acceptable after approval by a state orfederal regulatory agency or listing in the U.S. PharmacopeialConvention or other generally recognized sources for use in subjects.

The pharmaceutical carriers include any and all physiologicallycompatible solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike. Examples of pharmaceutical carriers include, but are not limitedto, sterile liquids, such as water, oils and lipids such as, forexample, phospholipids and glycolipids. These sterile liquids include,but are not limited to, those derived from petroleum, animal, vegetableor synthetic origin such as, for example, peanut oil, soybean oil,mineral oil, sesame oil, and the like. Water can be a preferred carrierfor intravenous administration. Saline solutions, aqueous dextrose andglycerol solutions can also be liquid carriers, particularly forinjectable solutions.

Suitable pharmaceutical excipients include, but are not limited to,starch, sugars, inert polymers, glucose, lactose, sucrose, gelatin,malt, rice, flour, chalk, silica gel, sodium stearate, glycerolmonostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol, and the like. The composition canalso contain minor amounts of wetting agents, emulsifying agents, pHbuffering agents, or a combination thereof. The compositions can takethe form of solutions, suspensions, emulsion, tablets, pills, capsules,powders, sustained-release formulations and the like. Oral formulationscan include standard carriers such as, for example, pharmaceuticalgrades mannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate, and the like. See Martin, E. W.Remington's Pharmaceutical Sciences. Supplementary active compounds canalso be incorporated into the compositions.

In some embodiments, the carrier is suitable for parenteraladministration. In other embodiments, the carrier can be suitable forintravenous, intraperitoneal, intramuscular, sublingual or oraladministration. In other embodiments, the pharmaceutically acceptablecarrier may comprise pharmaceutically acceptable salts.

Pharmaceutical formulations for parenteral administration may includeliposomes. Liposomes and emulsions are delivery vehicles or carriersthat are especially useful for hydrophobic drugs. Depending onbiological stability of the therapeutic reagent, additional strategiesfor protein stabilization may be employed. Furthermore, one mayadminister the drug in a targeted drug delivery system such as, forexample, in a liposome coated with target-specific antibody. Theliposomes can be designed, for example, to bind to a target protein andbe taken up selectively by the cell expressing the target protein.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable for a high drug concentration. In some embodiments, the carriercan be a solvent or dispersion medium including, but not limited to,water; ethanol; a polyol such as for example, glycerol, propyleneglycol, liquid polyethylene glycol, and the like; and, combinationsthereof. The proper fluidity can be maintained in a variety of ways suchas, for example, using a coating such as lecithin, maintaining arequired particle size in dispersions, and using surfactants.

In some embodiments, isotonic agents can be used such as, for example,sugars; polyalcohols that include, but are not limited to, mannitol,sorbitol, glycerol, and combinations thereof; and sodium chloride.Sustained absorption characteristics can be introduced into thecompositions by including agents that delay absorption such as, forexample, monostearate salts, gelatin, and slow release polymers.Carriers can be used to protect active compounds against rapid release,and such carriers include, but are not limited to, controlled releaseformulations in implants and microencapsulated delivery systems.Biodegradable and biocompatible polymers can be used such as, forexample, ethylene vinyl acetate, polyanhydrides, polyglycolic acid,collagen, polyorthoesters, polylactic acid, polycaprolactone,polyglycolic copolymer (PLG), and the like. Such formulations cangenerally be prepared using methods known to one of skill in the art.

The compounds may be administered as suspensions such as, for example,oily suspensions for injection. Lipophilic solvents or vehicles include,but are not limited to, fatty oils such as, for example, sesame oil;synthetic fatty acid esters, such as ethyl oleate or triglycerides; andliposomes. Suspensions that can be used for injection may also containsubstances that increase the viscosity of the suspension such as, forexample, sodium carboxymethyl cellulose, sorbitol, or dextran.Optionally, a suspension may contain stabilizers or agents that increasethe solubility of the compounds and allow for preparation of highlyconcentrated solutions.

In one embodiment, a sterile and injectable solution can be prepared byincorporating an effective amount of an active compound in a solventwith any one or any combination of desired additional ingredientsdescribed above, filtering, and then sterilizing the solution. Inanother embodiment, dispersions can be prepared by incorporating anactive compound into a sterile vehicle containing a dispersion mediumand any one or any combination of desired additional ingredientsdescribed above. Sterile powders can be prepared for use in sterile andinjectable solutions by vacuum drying, freeze-drying, or a combinationthereof, to yield a powder that can be comprised of the activeingredient and any desired additional ingredients. Moreover, theadditional ingredients can be from a separately prepared sterile andfiltered solution. In another embodiment, the extract may be prepared incombination with one or more additional compounds that enhance thesolubility of the extract.

In some embodiments, a therapeutically or prophylactically effectiveamount of a composition may range in concentration from about 0.001 nMto about 0.10 M; from about 0.001 nM to about 0.5 M; from about 0.01 nMto about 150 nM; from about 0.01 nM to about 500 μM; from about 0.01 nMto about 1000 nM, 0.001 μM to about 0.10 M; from about 0.001 μM to about0.5 M; from about 0.01 μM to about 150 μM; from about 0.01 μM to about500 μM; from about 0.01 μM to about 1000 nM, or any range therein. Insome embodiments, the compositions may be administered in an amountranging from about 0.001 mg/kg to about 500 mg/kg; from about 0.005mg/kg to about 400 mg/kg; from about 0.01 mg/kg to about 300 mg/kg; fromabout 0.01 mg/kg to about 250 mg/kg; from about 0.1 mg/kg to about 200mg/kg; from about 0.2 mg/kg to about 150 mg/kg; from about 0.4 mg/kg toabout 120 mg/kg; from about 0.15 mg/kg to about 100 mg/kg, from about0.15 mg/kg to about 50 mg/kg, from about 0.5 mg/kg to about 10 mg/kg, orany range therein, wherein a human subject is assumed to average about70 kg.

In some embodiments, the compounds can be administered by inhalationthrough an aerosol spray or a nebulizer that may include a suitablepropellant such as, for example, dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or acombination thereof. In one example, a dosage unit for a pressurizedaerosol may be delivered through a metering valve. In anotherembodiment, capsules and cartridges of gelatin, for example, may be usedin an inhaler and can be formulated to contain a powderized mix of thecompound with a suitable powder base such as, for example, starch orlactose.

The present invention encompasses sustained release formulations for theadministration of one or more agents. In some embodiments, the sustainedrelease formulations can reduce the dosage and/or frequency of theadministrations of such agents to a subject.

The compositions can be administered as a pharmaceutical formulation byinjection. In some embodiments, the formulation can comprise the extractin combination with an aqueous injectable excipient. Examples ofsuitable aqueous injectable excipients are well known to persons ofordinary skill in the art, and they, and the methods of formulating theformulations, may be found in such standard references as Alfonso A R:Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company,Easton Pa., 1985. Suitable aqueous injectable excipients include water,aqueous saline solution, aqueous dextrose solution, and the like,optionally containing dissolution enhancers for the acid-modifiedarabinogalactan protein composition, such as solution of mannitol orother sugars, or a solution of glycine or other amino acids.

Typically, a composition taught herein can be administered bysubcutaneously, intramuscularly, intraperitoneally, or intravenously,injecting. A localized administration can, in some embodiments, includedirect injection of an agent into the region of the tissue to be treatedsuch as, for example, a solid tumor. In some embodiments, intravenousadministration is used, and it can be continuous intravenous infusionover a period of a few minutes to an hour or more, such as aroundfifteen minutes. The amount administered may vary widely depending onthe type of formulation, size of a unit dosage, kind of excipients, andother factors well known to those of ordinary skill in the art. Theformulation may comprise, for example, from about 0.0001% to about 10%(w/w), from about 0.01% to about 1%, from about 0.1% to about 0.8%, orany range therein, with the remainder comprising the excipient orexcipients.

In some embodiments, the composition can be administered in conjunctionwith at least one other therapeutic agent for the disease state beingtreated, especially another agent capable of treating cancer such as,for example, a chemotherapeutic agent. The amounts of the agents neededcan be reduced, even substantially, such that the amount of the agent oragents required is reduced to the extent that a significant response isobserved from the subject. A significant response can include, but isnot limited to, a reduction or elimination of nausea, a visible increasein tolerance, a faster response to the treatment, a more selectiveresponse to the treatment, or a combination thereof.

The methods can further comprise the administration of an effectiveamount of an antiproliferative, an effective amount of radiationtherapy, surgical therapy, or a combination thereof. The teachings arealso directed to a method of treating a cancer. In some embodiments, themethod comprises administering an agent to a subject in need of a cancertreatment, wherein the dose of the agent is selected to reduce oreliminate an immunosuppression that would otherwise occur whenadministering a substantially higher dose of the agent in the subject;and administering radiation therapy in combination with the agent,wherein the reduction or elimination of the immunosuppression enhancesthe efficacy of the radiation therapy when compared to the efficacy ofthe radiation therapy otherwise observed when administered incombination with the substantially higher dose of the agent in thesubject. In some embodiments, the agent comprises one or morechemotherapeutic agents in combination with the agents provided herein.In these embodiments, the agent can be selected from the groupconsisting of dacarbazine, paclitaxel, doxorubicin, or a combinationthereof.

In some embodiments, an effective amount can range, for example, fromabout 1 mg/day to about 1000 mg/day, from about 10 mg/day to about 500mg/day, from about 50 mg/day to about 250 mg/day, or any range therein,for a human of average body mass. For treating a solid tumor, a similaramount will be therapeutically effective. A person of ordinary skill inthe art will be able without undue experimentation, having regard tothat skill and this disclosure, to determine a therapeutically effectiveamount of the compositions of this invention for a given disease.

In some embodiments, G-CSF is administered in combination with acomposition taught herein using any amount, time, and method ofadministration known to be effective by one of skill. The G-CSF can beNEUPOGEN, for example, administered in an amount ranging from about 0.1μg/kg to about 1 mg/kg, from about 0.5 μg/kg to about 500 μg/kg, fromabout 1 μg/kg to about 250 μg/kg, from about 1 μg/kg to about 100 μg/kgfrom about 1 μg/kg to about 50 μg/kg, or any range therein.

In some embodiments, the radiation therapy can be administered in asingle, localized high-dose ranging, for example, from about 20 Gy toabout 100 Gy. In some embodiments, the radiation therapy can beadministered in a total dose ranging from about 20 Gy to about 100 Gyusing a modified hypofractionation regime of dosing comprising fromabout 2 doses to about 5 doses during a time frame of one week. In someembodiments, the radiation therapy can be administered in a total doseranging from about 20 Gy to about 100 Gy using a modifiedhypofractionation regime of dosing comprising from 2 doses to 3 dosesduring a time frame ranging from about 2 days to about 3 days. Theradiation therapy can also be administered in a total dose ranging fromabout 45 Gy to about 60 Gy using a modified hypofractionation regime ofdosing comprising administering a single dose ranging from about 15 Gyto about 20 Gy for each day during a 3-day time frame.

The compositions and therapies taught herein can be administered incombination. For example, the combinations can be administered, forexample, for 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 18hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9days, 10 days, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 3 months, 6 months 1year, any combination thereof, or any amount of time considerednecessary by one of skill. The agents can be administered concomitantly,sequentially, or cyclically to a subject. Cycling therapy involves theadministering a first agent for a predetermined period of time,administering a second agent or therapy for a second predeterminedperiod of time, and repeating this cycling for any desired purpose suchas, for example, to enhance the efficacy of the treatment. The agentscan also be administered concurrently. The term “concurrently” is notlimited to the administration of agents at exactly the same time, butrather means that the agents can be administered in a sequence and timeinterval such that the agents can work together to provide additionalbenefit. Each agent can be administered separately or together in anyappropriate form using any appropriate means of administering the agentor agents.

Articles of Manufacture

The present invention provides for articles of manufacture thatencompass finished, packaged and labelled pharmaceutical products. Thearticles of manufacture include the appropriate unit dosage form in anappropriate vessel or container such as, for example, a glass vial orother container that is hermetically sealed. In the case of dosage formssuitable for parenteral administration, the active ingredient, e.g. oneor more agents including an extract taught herein, is sterile andsuitable for administration as a particulate-free solution. In otherwords, the invention encompasses both parenteral solutions andlyophilized powders, each being sterile, and the latter being suitablefor reconstitution prior to injection. Alternatively, the unit dosageform may be a solid suitable for oral, transdermal, topical or mucosaldelivery.

In some embodiments, the unit dosage form is suitable for intravenous,intramuscular, topical or subcutaneous delivery. Thus, the inventionencompasses solutions, which are preferably sterile and suitable foreach route of delivery. The concentration of agents and amountsdelivered are included as described herein.

As with any pharmaceutical product, the packaging material and containerare designed to protect the stability of the product during storage andshipment. In addition, the articles of manufacture can includeinstructions for use or other information material that can advise theuser such as, for example, a physician, technician or patient, regardinghow to properly administer the composition as a prophylactic,therapeutic, or ameliorative treatment of the disease of concern. Insome embodiments, instructions can indicate or suggest a dosing regimenthat includes, but is not limited to, actual doses and monitoringprocedures.

In other embodiments, the instructions can include informationalmaterial indicating that the administering of the compositions canresult in adverse reactions including but not limited to allergicreactions such as, for example, anaphylaxis. The informational materialcan indicate that allergic reactions may exhibit only as mild pruriticrashes or may be severe and include erythroderma, vasculitis,anaphylaxis, Steven-Johnson syndrome, and the like. The informationalmaterial should indicate that anaphylaxis can be fatal and may occurwhen any foreign protein is introduced into the body. The informationalmaterial should indicate that these allergic reactions can manifestthemselves as urticaria or a rash and develop into lethal systemicreactions and can occur soon after exposure such as, for example, within10 minutes. The informational material can further indicate that anallergic reaction may cause a subject to experience paresthesia,hypotension, laryngeal edema, mental status changes, facial orpharyngeal angioedema, airway obstruction, bronchospasm, urticaria andpruritus, serum sickness, arthritis, allergic nephritis,glomerulonephritis, temporal arthritis, eosinophilia, or a combinationthereof.

In some embodiments, the articles of manufacture can comprise one ormore packaging materials such as, for example, a box, bottle, tube,vial, container, sprayer, insufflator, intravenous (I.V.) bag, envelope,and the like; and at least one unit dosage form of an agent comprisingan extract taught herein within the packaging material. In otherembodiments, the articles of manufacture may also include instructionsfor using the composition as a prophylactic, therapeutic, orameliorative treatment for the disease of concern.

In other embodiments, the articles of manufacture can comprise one ormore packaging materials such as, for example, a box, bottle, tube,vial, container, sprayer, insufflator, intravenous (I.V.) bag, envelope,and the like; and a first composition comprising at least one unitdosage form of an agent comprising an extract as taught herein withinthe packaging material, along with a second composition comprising asecond agent such as, for example, a glycosaminoglycan, phospholipid,poly(alkylene glycol), any other bioactive agent taught herein, or anyprodrugs, codrugs, metabolites, analogs, homologues, congeners,derivatives, salts and combinations thereof. In other embodiments, thearticles of manufacture may also include instructions for using thecomposition as a diagnostic, prophylactic, therapeutic, or ameliorativetreatment for the disease of concern.

Without intending to be limited to any theory or mechanism of action,the following examples are provided to further illustrate the teachingspresented herein. It should be appreciated that there are severalvariations contemplated within the skill in the art, and that theexamples are not intended to be construed as providing limitations tothe claims.

EXAMPLE 1 A Human Model was Prepared to Correlate Angiogenesis with theExtracellular Matrix

In this study, we investigated the correlation between sproutangiogenesis and the integrity of an extracellular matrix (ECM)environment using in vivo and in vitro angiogenesis models. We used anαvβ3 antagonist and an α2β1 antagonist in the model, where the αvβ3antagonist was the disintegrin echistatin, and the α2β1 antagonist wasthe disintegrin VP12 (ECL12).

Materials

A 3-D ECM model was prepared. Gelatin-coated, microcarrier beads(Cytodex-3) were purchased from Pharmacia (Uppsala, Sweden). Sterile,native bovine dermal collagen containing 95% type I collagen and 5% typeIII collagen (Vitrogen) was obtained from Collagen Biomaterials (PaloAlto, Calif.). Dimethyl dichlorosilane, aprotinin, dibutyryl cyclic AMP,hydrocortisone, trypsin, soybean trypsin inhibitor, and EDTA wereobtained from Sigma Chemical Co. (St. Louis, Mo.). Endothelial cellbasal medium (EBM), endothelial cell growth medium bulletkit-2 (EGM-2BULLETKIT), bovine brain extract, and epidermal growth factor wereobtained from Clonetics Corp. (San Diego, Calif.). Normal human serumwas obtained from BioWhittaker, Inc. (Walkersville, Md.). Plateletderived growth factor-BB (PDGF-BB), vascular endothelial cell growthfactor (VEGF) and Basic fibroblast growth factor (bFGF) were purchasedfrom Chemical Co. (St. Louis, Mo.). VEGF-C was kindly provided by KariAlitalo. Human thrombin was obtained from Calbiochem (San Diego,Calif.). And, propidium iodide (PI) was obtained from Molecular Probes(Eugene, Oreg.).

Cell Culture

Human dermal microvascular endothelial cells (HDMEC) were isolated fromhuman neonatal foreskins. Briefly, after initial harvest from mincedtrypsinized human foreskins, microvascular endothelial cells werefurther purified on a PERCOLL density gradient. HDMEC were cultured oncollagen type 1 coated tissue culture flasks in EGM (endothelial cellgrowth medium) consisting of EBM supplemented with 10 ng/ml epidermalgrowth factor, 0.4% bovine brain extract, 17.5 microg/ml dibutyrylcyclic AMP, and 1 microg/ml hydrocortisone in the presence of 30% normalhuman serum. Endothelial cell cultures were characterized and determinedto be >99% pure on the basis of formation of typical cobblestonemonolayers in culture, positive immunostaining for factor VIII-relatedantigen, and selective uptake of acetylated low density lipoprotein. Allexperiments were done with HDMEC below passage 10. Bovine aorticendothelial cells (BAEC) were obtained from BioWhittaker, Inc.(Walkersville, Md.) and cultured according to manufacturer'sinstruction. BAEC between passage 4 and 8 were used for experiments.

Preparation of Endothelial Cell-Loaded Microcarrier Beads (EC-Beads)

Gelatin-coated CYTODEX-3 microcarrier beads were prepared as describedby the manufacturer. Approximately 80,000 sterile microcarrier beadswere washed, resuspended in EGM, and added to approximately 4.5 millionendothelial cells (HDMEC or BAEC). The beads and cells were mixed bygentle swirling, incubated at 37° C. for 6 hr, and then rotated for24-36 hr on an orbital mixer in a 37° C. oven to generate endothelialcell-loaded microcarrier beads (EC-beads).

EXAMPLE 2 Cell Migration and Capillary Sprout Formation was Identifiedin Fibrin Gels and Type I Collagen Gels in the Human Model

FIGS. 1A-1C illustrate a study of human microvascular endothelial cellangiogenesis, according to some embodiments. A microcarrier, in vitroangiogenesis assay, previously designed to investigate bovine pulmonaryartery endothelial cell angiogenic behavior in bovine fibrin gels, wasmodified for the study of human microvascular endothelial cellangiogenesis. The HDMEC were isolated from human neonatal foreskins andused, as described above, and images were captured at variousmagnifications, where the effect of angiogenic factors on sproutangiogenesis was quantified visually by counting the number and percentof EC-beads with capillary sprouts.

FIG. 1A shows the process in Step I, where human fibrinogen, isolated aspreviously described, was dissolved in M199 medium at a concentration of1 mg/ml (pH 7.4) and sterilized by filtering through a 0.22 micronfilter. An isotonic 1.5 mg/ml collagen solution was prepared by mixingsterile native bovine type I &III collagen (Vitrogen, CollagenBiomaterials, Palo Alto, Calif.) in 5×M199 medium and distilled water.The pH was adjusted to 7.4 using 1N NaOH.

FIG. 1A also shows the process in Step II, where in certain experiments,growth factors, such as VEGF, VEGF-C, bFGF or PDGF-BB, were added to thefibrinogen and collagen solutions.

For experiments using RGD peptides, EC-beads were firstly incubated withvary concentrations of peptides for one hour and then were added to theECM solutions. About 500 EC-beads were then added to the ECM proteinsolutions, followed by the addition of 0.5 U/ml human thrombin. A 0.3 mlaliquot of each suspension was immediately added to appropriate wells ofa 24-well tissue culture plate. After gelation, 1 ml of fresh assaymedium (EBM supplemented with 20% normal human serum for HDMEC or EBMsupplemented with 10% fetal bovine serum for BAEC) was added to eachwell.

FIGS. 1B and 1C show how the angiogenic response was monitored visuallyand recorded by video image capture. Specifically, capillary sproutformation was observed and recorded with a NIKON Diaphot-TMD invertedmicroscope (Nikon Inc., Melville, N.Y.), equipped with an incubatorhousing with a NIKON NP-2 thermostat and Sheldon #2004 carbon dioxideflow mixer. The microscope was directly interfaced to a video systemconsisting of a Dage-MTI CCD-72S video camera and Sony 12″ PVM-122 videomonitor linked to a Macintosh G3 computer. The images were captured atvarious magnifications using Adobe Photoshop. The effect of angiogenicfactors on sprout angiogenesis was quantified visually by determiningthe number and percent of EC-beads with capillary sprouts. 100-200 beads(five random low power fields) in each of triplicate wells were countedfor each experimental condition. All experiments were repeated at leastthree times. FIG. 1B shows an absence of sprout angiogenesis usinganti-angiogenic factor, and FIG. 1C shows the presence of sproutangiogenesis using angiogenic factor.

FIGS. 2A-2D show the HDMEC of the model with angiogenesis formation,according to some embodiments. FIGS. 2A and 2B show the HDMEC nucleusand vessels. FIGS. 2C and 2D show a vessel formation and a lumen in thevessels using reflective, confocal microscopy. To locate the nucleus ofHDMEC, the fibrin or collagen gel were fixed by methanol/acetone (1:1)and stained by 0.001% PI.

EXAMPLE 3 Integrin Receptors were Identified Using Porcine CutaneousWounds and Immunofluorescence Staining

Porcine cutaneous wounds were harvested at various times and thenimmunoprobed for expression of integrin receptors. See Xu, J and Clark,R. The Journal of Cell Biology 132:239-249(1996). Briefly,full-thickness wounds were made with an 8-mm punch on the backs of WhiteYorkshire pigs and harvested at the times indicated. Specimens werebisected; one half was fixed in formalin and stained with MASSONtrichrome, the other half was frozen in liquid nitrogen forimmunofluorescence studies. Anti-laminin antibodies (Gibco BRL)conjugated with biotin were used to identify wound vasculature. Allantibodies were used at dilutions that gave maximal specificfluorescence and minimal background fluorescence on frozen tissuespecimens. Bound antibody was detected by the avidin-biotin-complex(ABC) technique. Stained specimens were observed and photographed usinga NIKON Microphot FXA epifluorescence microscope equipped with a NIKONFX-35DX 35mm camera.

EXAMPLE 4 A Positive Correlation was Identified Between WoundAngiogenesis and a Provisional Matrix

To understand the relationship between angiogenesis and different ECMcomponents during granulation tissue formation of wound repair, weanalyzed tissue specimens from 5, 7 and 10 day porcine wounds. Atransition from a fibrin-rich provisional matrix to a substantiallyorganized collagen fiber network was observed, where the scarring of thecollagen fiber network caused vasculature to regress.

FIGS. 3A-3F show would repair in 5, 7, and 10 day porcine wounds,according to some embodiments. The 5 day wounds were mainly composed ofa fibrin-rich provisional matrix, whereas the 7 day wounds had asubstantially organized collagen fiber network. The 10 day wounds haddeveloped a compacted, contracted collagen scar.

In FIG. 3, the staining of 5, 7, and 10 day wound specimens withanti-laminin shows the transition from a fibrin-rich provisional matrixto a substantially organized collagen fiber network, and its effects onangiogenesis. As shown in FIGS. 3A and 3D, for example, the fibrin-richearly granulation tissue in 5 day wounds is filled with newly formedvessels. As shown in FIGS. 3B and 3E, however, the maturing bloodvessels form an organized vertical array in about 7 days as collagenaccumulates in the wound ECM. As shown in FIGS. 3C and 3F, the collagenbundles thicken to produce scar at about 10 days, and many blood vesselsare regressing. Thus, during wound repair in vivo, the angiogenicneovessels in early granulation tissue mature and then regress as fibrinis replaced by collagen in the wound space. FIGS. 4A-4F show thestaining of 5, 7, and 10 day wound specimens with Masson trichrome,according to some embodiments. The results shown in FIG. 4 correlatewell with the results shown in FIG. 3. The data suggests that collagenand fibrin differentially, and synergistically, regulate sproutangiogenesis.

EXAMPLE 5 Fibrin Supports Sprouting Angiogenesis

FIGS. 5A-5E show that fibrin supports sprouting angiogenesis, accordingto some embodiments. In our 3-D fibrin gel model, with stimulation ofangiogenic stimulators (30 ng VEGF+25 ng/ml bFGF), the HDMEC formedcapillary sprouts. FIG. 5A is the control having no angiogenicstimulators, and in which no significant HDMEC sprout formationoccurred. FIG. 5B shows projections of the sprouts from the surface ofthe EC-beads, which invaded into the fibrin gel within 48 hrs. FIG. 5Cshows that, by 5 days, the endothelial sprouts had elongated, and insome cases, formed branching capillary sprouts. FIG. 5D show that localcapillary networks formed by branching and fusion of capillary sproutsfrom the same bead. FIGS. 5E and 5F show that wild capillary networksformed by fusion of capillary sprouts from adjacent beads.

EXAMPLE 6 Collagen Does Not Support Sprouting Angiogenesis

Using an in vitro three dimensional microcarrier based sproutangiogenesis system, we further demonstrated that fibrin and type Icollagen 3-D matrix differentially regulate angiogenic sprout formationof HDEMC. FIGS. 6A-6H show that fibrin supports sprouting angiogenesiswhere collagen does not support sprouting angiogenesis, according tosome embodiments. FIGS. 6A, 6C 6,E, and 6G show that, in the presence ofVEGF in fibrin gel, HDMEC formed capillary-like sprouts from the surfaceof EC-beads and invaded and migrated into the surrounding fibrin. Incontrast, FIGS. 6B, 6D 6,F, and 6H show that when VEGF was added tocollagen gel, HDMEC invaded and migrated into the surrounding collagenas individual cells, but did not form sprouts.

EXAMPLE 7 Fibrin and Collagen Receptors Synergistically RegulateAngiogenesis

We've shown that integrin β3 expression is highly up-regulated in fibrinrich, but not in collagen rich, matrix environments in vitro and invivo. The 3D HDMEC model described above was used in this experimentthat demonstrates combination therapy using two disintegrins, echistatinand VP12 (ECL12).

Disintegrins represent a novel family of integrin β1 and β3 inhibitorproteins isolated from viper venoms. They are low molecular-weight,cysteine-rich peptides containing the Arg-Gly-Asp (RGD) sequence. Theyare the most potent known inhibitors of integrin function. Disintegrinsinterfere with cell adhesion to the extracellular matrix, includingadhesion of melanoma cells and fibroblasts to fibronectin, and arepotent inhibitors of platelet aggregation.

Echistatin (Disintegrin echistatin-alpha or Carinatin) is derived fromthe venom of Echis carinatus (Saw-scaled viper). It is a potentirreversible αvβ3 integrin antagonist that disrupts attachment ofosteoclasts to bone and inhibits bone reabsorption, prevents ADP-inducedplatelet aggregation via inhibition of glycoprotein IIb/IIIa(GpIIb/IIIa, αIIbβ3) receptors. Echistatin inhibits fibrinogeninteraction with platelet receptors expressed on the glycoproteinIIb-IIIa complex, acts by binding to the glycoprotein IIb-IIIa receptoron the platelet surface, and inhibits aggregation induced by ADP,thrombin, platelet-activating factor and collagen. The followingstructure is a representation of echastatin, a 49 amino acid structurefor echistatin:

(SEQ ID NO: 1) 1        10         20         30         40QCESGPCCRN CKFLKEGTIC KRARGDDMDD YCNGKTCDCP         49 RNPHKGPAT

Echistatin is commercially available, for example, from Sigma-aldrich.See CAS Nos. 129038-42-2 and 154303-05-6. See also, Calvete, J. Biochem.J. 372:725-734(2003), which is hereby incorporated by reference hereinin its entirety.

VP12 (ECL12) (Vipera paleastinae venom or VP12) isolated from Viperapaleastinae venom showed a potent inhibitory activity against collagenreceptors α2β1 integrins. Structurally, VP12 is composed of two subunitsVP12A and VP12B displaying amino acid sequence homology withheterodimeric C-lectin type proteins. Sigma Aldrich offers Viperapaleastinae venom under catalog no. V0628, and the composition used forthis example was graciously received from Dr. Cezary Marcinkiewicz,Biotechnology Center, Temple University College of Science andTechnology, Philadelphia, Pa. 19122. See Cancer Biol Ther.8(15):1507-16(2009), which is hereby incorporated by reference herein inits entirety.

(SEQ ID NO:2)

The VP12 (ECL12) subunits VP12A and VP12B are shown, where the grayareas represent conserved amino acids, gaps (−) were included tomaximize sequence similarities, and the X represents unidentified aminoacids in the VP12A subunit. See also, Staniszewska, I. Cancer Biology &Therapy 8(15):1507-1516(2009), which is hereby incorporated by referenceherein in its entirety.

FIGS. 7A-7D show synergistic inhibition of sprout angiogenesis whencombining echistatin, an αvβ3 antagonist with VP12 (ECL12), an α2β1agonist, according to some embodiments. FIG. 7A shows sproutangiogenesis in a HDMEC culture in VEGF and bFGF. FIG. 7B shows theinhibition of sprout angiogenesis when echistatin is added to theculture of FIG. 7A. FIG. 7C shows the inhibition of sprout angiogenesiswhen VP12 (ECL12) is added to the culture of FIG. 7A. FIG. 7D, however,shows the synergistic inhibition of sprout angiogenesis when bothechistatin and VP12 (ECL12) are added to the culture of FIG. 7A.

Echistatin, a disintegrin specific for αvβ3, dose dependently inhibitssprout angiogenesis of HDMEC in fibrin. While VP12 (ECL12), adisintegrin inhibitor to collagen receptor integrin α2β1, has noinhibitory effect on sprout angiogenesis in vitro. Surprisingly,however, this example illustrates how VP12 (ECL12) significantly andsynergistically enhanced the inhibition of sprout angiogenesis byechistatin when administered in combination with echistatin.

EXAMPLE 8 A Combination of Echistatin and Blocking mAb for α2β1

An experiment similar to Example 7 was designed to replace VP12 (ECL12)with a blocking antibody for α2β1. The echistatin, a disintegrinspecific for αvβ3, dose dependently inhibited sprout angiogenesis ofHDMEC in fibrin. Again, surprisingly, while the blocking antibody tointegrin α2β1, a receptor for collagen, had no inhibitory effect onsprout angiogenesis in vitro, it synergistically enhanced the inhibitionof sprout angiogenesis by echistatin.

In this experiment, JBS2, a blocking mab for integrin α2β1, waspurchased from Chemicon (Temecula, Calif.). JBS2 showed no significantinhibitory effect on sprout angiogenesis of HDMEC in the 3D fibrinmatrix. Echistatin, a disintegrin specific for αvβ3, dose dependently (1μg/ml to 10 μg/ml) inhibited sprout angiogenesis of HDMEC in fibrin. Atthe concentration of 0.1 μg/ml, echistatin showed little inhibitoryeffect on sprout angiogenesis of HDMEC in fibrin. However, when combinedwith JBS2, which has no significant inhibitory effect on sproutangiogenesis in fibrin, 0.1 μg/ml echistatin totally inhibited sproutangiogenesis in fibrin gel. It suggests that blocking integrin α2β1synergistically enhanced the inhibitory effect of αvβ3 for sproutangiogenesis. It indicates that blocking integrin α2β1 significantlydeceased the effective dose of αvβ3 antagonists to inhibit sproutangiogenesis in fibrin. Furthermore, blocking integrin α2β1 enhanced theinhibitory effect of αvβ3 antagonists on sprout angiogenesis in fibrin.

EXAMPLE 9 A Combination of an RGD Peptide and Blocking mAb for α2β1

This experiment was designed to replace echistatin with a blockingpeptide construct that binds to αvβ3 to determine how the VP12 (ECL12)worked in combination with other blocking peptides for αvβ3. A cyclicRGD peptide, cyclo (Arg-Gly-Asp-d-Phe-Cys),(cRGDfC), was purchased fromPeptides International (Louisville, Ky.) and used in this experiment.

The cyclic RGD peptide recognizes αvβ3 integrin receptors, which play apivotal role in angiogenesis, and the proliferation of malignant tumors.The cRGDfC dose dependently inhibited sprout angiogenesis of HDMEC in afibrin matrix. The blocking mAb for integrin α2β1 had no significanteffect on sprout angiogenesis of HDMEC in the fibrin matrix.

The integrin α2β1 is blocked to significantly enhance the effect ofblocking the integrin αvβ3 on angiogenesis and tumor invasion. Ablocking mAb of α2β1 will, therefore, be used to enhance, perhapssynergistically, the inhibitory effect of cRGDfC on angiogenesis andtumor invasion in a fibrin matrix, providing further evidence that thecombination of blocking integrin α2β1 with the blocking of integrin αvβ3significantly enhances the inhibitory effect of αvβ3 antagonists onangiogenesis and tumor invasion.

EXAMPLE 10 Liposome Formulation

One of skill will be able to readily prepare formulations foradministration, and liposomes may be desired. Liposomes may be preparedusing the following procedure. Different ratios of high transitiontemperature lipids, cholesterol, and lipids derivatized with long-chainpolymers (polyethylene glycol) can be used. The particles can be formedusing probe sonication. See Fujii, G. et al. Biochemistry36:4959-68(1997). A mixture of lipids, such asdisteroylphosphatidylcholine, cholesterol, and polyethylene glycolderivatized lipid, can be dissolved in a chloroform/methanol solution.Thin lipid films can be created by pipetting aliquots of the lipidsolutions into round-bottomed glass tubes and evaporating the solvent at65° C. under a stream of nitrogen gas. The films are placed under vacuumfor at least 24 h to remove residual organic solvent. Liposomes can beprepared by hydrating the lipid films with a disintegrin dissolved in 10mM sodium phosphate and 9% sucrose (pH 7.2) and then incubating thesuspension at 65° C. for 5-10 min followed by probe sonication until thesuspension is translucent. In the resultant suspension, unencapsulateddisintegrin is removed by ultrafiltration and the suspension issterilized prior to use by passage through a 0.22 Am filter. Theentrapped protein concentration can then be determined by disruption ofthe liposomes with chloroform/methanol/water (10:40:50) followed bycentrifugation at 14,000×g. The supernatant can be analyzed fordisintegrin concentration using the BCA assay (Pierce Chemical Co.,Rockford, Ill.) following an established protocol. See Smith, P K, etal. Anal Biochem 150:76-85(1985).

1. A pharmaceutical formulation comprising an αvβ3 antagonist, an α2β1antagonist, and a pharmaceutically acceptable carrier.
 2. Thepharmaceutical formulation of claim 1, comprising echistatin and VP12(ECL12).
 3. An article of manufacture comprising an αvβ3 antagonist, anα2β1 antagonist, and instructions for administering an effective amountof the αvβ3 antagonist and an effective amount of the α2β1 antagonist toa subject.
 4. A method of inhibiting angiogenesis in a subject,comprising administering an effective amount of an αvβ3 antagonist incombination with an effective amount of an α2β1 antagonist to thesubject.
 5. The method of claim 4, comprising administering echistatinand VP12 (ECL12) to the subject.
 6. The method of claim 4, wherein themethod further inhibits tumor invasion.
 7. The method of claim 4,wherein the method inhibits the growth of solid tumors.
 8. The method ofclaim 4, further comprising the administration of an effective amount ofan antiproliferative.
 9. The method of claim 4, further comprising theadministration of an effective amount of radiation therapy.
 10. A methodof inhibiting angiogenesis in a subject, comprising administering aneffective amount of echistatin in combination with an effective amountof VP12 (ECL12) to the subject.
 11. The method of claim 10, wherein themethod further inhibits tumor invasion.
 12. The method of claim 10,wherein the method further inhibits the growth of solid tumors.
 13. Themethod of claim 10, further comprising the administration of aneffective amount of an antiproliferative.
 14. The method of claim 10,further comprising the administration of an effective amount ofradiation therapy.
 15. A method of inhibiting the growth of a solidtumor in a subject, comprising administering an effective amount ofechistatin in combination with an effective amount of VP12 (ECL12) tothe subject.
 16. The method of claim 15, wherein the method furtherinhibits tumor invasion.
 17. The method of claim 15, further comprisingthe administration of an effective amount of an antiproliferative. 18.The method of claim 15, further comprising the administration of aneffective amount of radiation therapy.
 19. A method of inhibiting tumorinvasion in a subject, comprising administering an effective amount ofechistatin in combination with an effective amount of VP12 (ECL12) tothe subject.
 20. The method of claim 19, wherein the method inhibits thegrowth of solid tumors.
 21. The method of claim 19, further comprisingthe administration of an effective amount of an antiproliferative. 22.The method of claim 19, further comprising the administration of aneffective amount of radiation therapy.